SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin

Abstract

Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors1,2. Phagocytosis by macrophages plays a critical role in cancer control3,4,5,6. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo7,8,9,10, suggesting that blockade of the SIRPα–CD47 checkpoint could be useful in treating human cancer11,12,13,14. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα–CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα–CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions15,16,17, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs21,22. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα–CD47 blockade therapy.

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Figure 1: Macrophages phagocytose a subset of haematopoietic cells.
Figure 2: SLAM receptors are required for phagocytosis of haematopoietic cells.
Figure 3: SLAMF7 is necessary and sufficient for phagocytosis of haematopoietic cells.
Figure 4: SLAMF7 controls actin polarization and promotes phagocytosis independently of SAP adaptors.
Figure 5: SLAMF7-dependent phagocytosis requires ITAMs and Mac-1.

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Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MT-14429, MOP-82906, FDN-143338) and the Canadian Cancer Society Research Institute (CCSRI; grant 018114) to A.V., RO1AI65495, RO1AI68150 and RO1AI113272 from the National Institutes of Health to C.A.L., and the Natural Sciences and Engineering Research Council (NSERC 1050319) to J.S.D. J.C. is recipient of a Fellowship from the Cole Foundation, while S.M. is a fellowship recipient of the RDV Foundation and N.W. was recipient of a Fellowship from Fonds de la recherche du Québec – Santé (FRQ-S). D.C.V. is a Chercheur-clinicien boursier of FRQ-S. J.S.D. holds the Anne and Max Tanenbaum Chair in Molecular Medicine, University of Toronto. A.V. holds the Canada Research Chair in Signaling in the Immune System.

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Authors

Contributions

J.C. planned and performed most experiments, interpreted data, and wrote the manuscript. M.-C.Z. planned and performed mouse tumour formation experiments (with J.C.), and interpreted data. M.-C.Z., H.G., and D.D. together confirmed key results of phagocytosis assays, and interpreted data. H.G. performed the assays of anti-CD47 effects on calcium fluxes and protein tyrosine phosphorylation. D.D. performed the microscopy-based conjugate formation assay (with J.C.). H.G., Y.L., and L.-A.P.-Q. generated and characterized reagents, and interpreted data. S.M., I.R., S.Z., M.-E.C.-M., N.W., D.C.V., and M.S. characterized reagents and interpreted data. V.C. performed the bioinformatics analyses of SLAMF7 and CD47 expression. C.A.L. and J.S.D. planned experiments, interpreted data, revised the manuscript, and provided funding. A.V. planned experiments, generated reagents, interpreted data, wrote the manuscript, and obtained funding. All authors commented on the manuscript.

Corresponding author

Correspondence to André Veillette.

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Competing interests

Part of A.V.’s work (distinct from the work reported herein) is supported by a grant from Bristol-Myers Squibb to study the mechanism of action of elotuzumab, which targets SLAMF7, in multiple myeloma. A.V. and J.C. have filed a patent on SLAMF7 in SIRPα–CD47 checkpoint blockade therapy. J.S.D. is an inventor on filed and awarded patents using SIRPα-protein therapeutics for treatment of haematological malignancy.

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Reviewer Information Nature thanks C. Miller and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Phagocytosis assays and direct impact of anti-CD47 on target cells.

a, b, Same as Fig. 1b, except phagocytosis assessed by flow cytometry using Tac-expressing L1210 (a) or pHrodo dye (b). Mϕs, macrophages. c, Cell death (in the absence of added macrophages) was examined by staining with annexin V and propidium iodide (PI), and flow cytometry. d, Cell proliferation was studied by CFSE dilution and flow cytometry. MFI, mean fluorescence intensity. e, Ca2+ fluxes were analysed using the Ca2+ indicator dye Indo-1, and flow cytometry. Ionomycin served as positive control. Time of addition of stimuli is shown by arrow. f, Protein tyrosine phosphorylation was detected by anti-phosphotyrosine (pTyr) immunoblotting. Representative of four (a), three (bd), and two (e, f) independent experiments. Uncropped blots can be seen in Supplementary Fig. 1.

Extended Data Figure 2 Cell-surface receptors on target cells.

a, Expression of CD47 (blue lines); prefixes m, mouse; h, human. Filled curves, isotype controls. b, c, Expression of LRP-1 in BMDMs from LRP-1 KO mice (Lrp1fl/fl;Lys2-Cre) and mice expressing Lys2-Cre alone (as control) was verified by immunoblot (b), while phagocytosis was determined as detailed for Fig. 1d (c). *P < 0.05; **P < 0.01 (two-tailed Student’s t-tests). Results pooled from a total of three (L1210) and two (P815) independent mice (c). Each symbol represents one mouse. All data are means ± s.e.m. Flow cytometry profiles are representative of five (L1210, P815, CB17-3A8, WEHI-3, SP2/0, activated CD4+ T cells, Raji, Daudi), three (MEL, BI-141, EL-4, RMA-S, YAC-1, BW5147.3, B16, CMT-93, L929, thymocytes, resting CD4+ T cells, resting B cells, activated B cells), and two (SW480, SW620, Colo205) experiments (a). Immunoblots are representative of one experiment (deletion of the LRP-1-encoding gene was shown by genotyping in three experiments (data not shown)) (b). Uncropped blots can be seen in Supplementary Fig. 1.

Extended Data Figure 3 SLAM receptors are required for phagocytosis of haematopoietic cells, but not of other targets.

a, Expression of various cell surface markers, including SLAM receptors (blue lines). Filled curves, isotype controls. b, Phagocytosis analysed as detailed for Extended Data Fig. 1a, b, using a flow cytometry-based assay (top) or the pHrodo-based assay (bottom). Left, representative experiments; right, quantification. c, Same as Fig. 1d, using peritoneal macrophages. d, Expression of CD47 (blue lines) on parental and CD47 KO L1210 cells. Filled curves, isotype controls. e, Phagocytosis of IgG-containing immune complexes (I.C.), GFP-expressing E. coli, or IgG-opsonized sRBCs was examined by flow cytometry (blue lines). Filled curves, BMDMs in the absence of phagocytosis. Phagocytosis of apoptotic thymocytes was analysed using microscopy-based assay. f, Phagocytosis of RBCs from WT or CD47 KO mice (mRBCs) was analysed by microscopy. g, Phagocytosis of IgG-opsonized L1210 was analysed. *P < 0.05; **P < 0.01 (two-tailed Student’s t-tests). Results pooled from a total of four (top) and three (bottom) (b), three (left) and two (right) (c), three (e, g), and two (f) mice in independent experiments. Each symbol represents one mouse. All data are means ± s.e.m. Flow cytometry profiles are representative of four (a) and three (d; e, left) independent experiments.

Extended Data Figure 4 Tumour transplantation assays.

a, b, From the experiment depicted in Fig. 2e. Cells were analysed by flow cytometry, in the presence of a fixed number of fluorescent beads to allow quantitation of total cell numbers. Beads are boxed in R1, while L1210 are boxed in R2 (a). Numbers of peritoneal macrophages were determined (b). c, Schematic representation of the experiment presented in Fig. 2f. TG, thioglycollate. d, e, From experiment depicted in Fig. 2f. Cells were analysed as specified for a and b. f, g, Tumours from experiment depicted in Fig. 2g were dissected, weighed, measured, and analysed by flow cytometry. Two RAG-1 KO mice treated with anti-CD47 (mice 9 and 10) showed no clinically detectable tumour when alive. However, upon dissection, small nodules with no detectable mass on the scale were present. These nodules were processed and analysed as for the other tumours. Masses are denoted as ‘0’ in the Source Data. L1210 were GFP+; macrophages were Ly6GCD11b+NK1.1; neutrophils were Ly6G+CD11b+NK1.1; and NK cells were Ly6GCD11b+NK1.1+. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student’s t-tests). Results pooled from a total of six mice analysed in five independent experiments (b), two mice (e), or 11 mice in two of four independent experiments (f, g). Each symbol represents one mouse. All data are means ± s.e.m. Dot plots are representative of six (a) or two (d) independent mice. See Source Data. Source data

Extended Data Figure 5 Impact of SLAMF7 on phagocytosis.

a, Expression of various cell surface markers, including SLAMF7 (blue lines). Filled curves, isotype controls. b, Phagocytosis was tested as detailed in Fig. 1d. For human targets (Raji, Daudi), F(ab′)2 fragments of antibodies were used. c, BAC transgenic mice expressing SLAMF7 were generated as detailed in Methods. In brief, the C57BL/6 BAC clone was first truncated at the 3′ end to eliminate the Slamf1 gene. Then, a stop codon (denoted by ‘X’) was introduced in exon 2 of Slamf2, the gene coding for CD48, and a silent mutation (HindIII site; denoted by short vertical red line) was created in Slamf7 to allow screening of transgenic mice. The transcriptional orientation of the Slam genes is depicted by arrows, while the relative positions of the genes in the clone are indicated by their distances from the 5′ end (in kilobases). d, Expression of various cell surface markers, including SLAMF7 (blue lines). Filled curves, isotype controls. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student’s t-tests). Results pooled from a total of four mice studied in independent experiments (b). Each symbol represents one mouse. All data are means ± s.e.m. Flow cytometry profiles are representative of four (a) and three (d) independent experiments.

Extended Data Figure 6 Expression of SLAMF7 on target cells.

a, b, Expression of various cell surface markers, including SFRs and their ligands (blue lines); prefixes m, mouse; h, human. Filled curves, isotype controls. c, Phagocytosis of L1210 by SFR KO macrophages expressing GFP alone or with SLAMF7 (human or mouse). Left, representative flow cytometry analysis; right, quantification. d, Phagocytosis of L1210 by macrophages from WT C57BL/6 or NRG mice, in the presence of anti-mSLAMF7 monoclonal antibody 4G2 or control rat IgG. Results pooled from a total of three (c), and three (C57BL/6) or two (NRG) (d) independent mice. Each symbol represents one mouse. All data are means ± s.e.m. Flow cytometry profiles are representative of three (MEL, BI-141, EL-4, RMA-S, YAC-1, BW5147.3, B16, CMT-93, L929, thymocytes, resting CD4+ T cells, resting B cells, activated B cells) or two (SW480, SW620, Colo205) (a), and four (b) independent experiments.

Extended Data Figure 7 Mechanism of action of SLAMF7.

a, Formation of conjugates (boxed) between macrophages and L1210 was detected by flow cytometry. Left, representative experiment. The percentages of conjugate formation are indicated above the boxes. Right, quantification. b, Same as Fig. 1d, using EAT-2 KO macrophages and L1210. c, Same as Fig. 1d, using WT or SFR KO macrophages and L1210, in the presence of kinase inhibitors. d, e, Expression of various cell surface markers (blue lines); filled curves, control antibodies. Phagocytosis of IgG-opsonized L1210 was analysed as detailed for Extended Data Fig. 3g. *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed Student’s t-tests). Results pooled from a total of three (ac) or two (d, e) mice studied in independent experiments. Each symbol represents one mouse. All data are means ± s.e.m. Flow cytometry profiles representative of two independent experiments (d, left; e, left).

Extended Data Figure 8 Impact of FcRγ and DAP12 on phagocytosis.

ae, Same as Fig. 1d and Extended Data Fig. 3g, using macrophages from DAP12 KO (a, b), FcRγ KO (c, d), or dKO (e) mice and L1210. a, c, Top, representative anti-DAP12 and anti-FcRγ immunoblots. **P < 0.01; ***P < 0.001 (two-tailed Student’s t-tests). Results pooled from a total of three (a, bottom), two (b, bottom; d, right; e, right), and five (c, bottom) mice studied in independent experiments. Each symbol represents one mouse. All data are means ± s.e.m. Flow cytometry profiles are representative of two independent experiments (b, top; d, left; e, left). Uncropped blots can be seen in Supplementary Fig. 1.

Extended Data Figure 9 Influence of integrins on phagocytosis.

a, Mass spectrometry analysis of anti-SLAMF7 immunoprecipitates. The GenInfo Identifier (gi) accession number and means of the normalized total ion current (TIC) for the potential interactors are shown. b, Same as a, except that CD11b was immunoprecipitated. c, Same as b, except that the data for FcRs CD64 and CD16 are shown. d, Expression of SLAMF7 and Flag (blue lines); filled curves, isotype control antibodies. e, Phagocytosis of L1210 was analysed as detailed for Fig. 1d. f, Expression of various cell surface markers (blue lines); filled curves, isotype control antibodies. g, h, Phagocytosis of L1210 opsonized with C3bi or IgG was analysed as detailed for Extended Data Fig. 3g. *P < 0.05; **P < 0.01 (two-tailed Student’s t-tests). Results pooled from two experiments with a total of five (a) or six (b, c) immunoprecipitates from independent mice, or three (e, g, h) independent experiments. Each symbol represents one mouse. All data are means ± s.e.m. Flow cytometry profiles are representative of four (d) or three (f) independent experiments.

Extended Data Figure 10 Gene expression analyses of SLAMF7 and CD47.

Expression of SLAMF7 and CD47 RNA in human haematological tumours. a, RNA levels of SLAMF7 (top) and CD47 (bottom) in several types and subtypes of leukaemia were analysed, using data obtained from microarray experiments. Data for only one oligonucleotide probe are shown. However, similar findings were made with other SLAMF7 and CD47 probes (data not shown). Each symbol represents a different patient sample. Median expression for a given type or subtype of malignancy is depicted by a horizontal line. For statistical analysis, Student’s t-tests were performed comparing SLAMF7 expression in the combination of all acute myelogenous leukaemia (AML) and acute lymphocytic leukaemia (ALL), versus either myelodysplastic syndrome (MDS) or chronic lymphocytic leukaemia (CLL). CML, chronic myelogenous leukaemia. b, Same as a, except that samples of multiple myeloma (MM) were analysed. c, Same as a, except that samples of acute myelogenous leukaemia and diffuse large B-cell lymphoma (DLBCL) were studied. Moreover, RNA expression was quantitated by RNA sequencing. d, Levels of SLAMF7 and CD47 RNAs for individual samples from selected tumour types, which displayed higher levels of SLAMF7 RNA, were analysed in parallel using dot plots. ***P < 0.001. The values of n, from left to right, are (a) MILE Study: 38, 41, 37, 28, 48, 352, 70, 237, 122, 13, 40, 36, 58, 174, 206, 76, 448; AML TCGA: 4, 20, 16, 91, 27, 6, 14, 1, 14, 3, 17, 3, 5, 7, 7, 6, 1, 2; (b) multiple myeloma: 304; (c) TCGA AML: 173; TCGA diffuse large B-cell lymphoma: 48; (d) 13, 206, 448, 20, 14, 17, 304, 173, 48.

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Chen, J., Zhong, M., Guo, H. et al. SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin. Nature 544, 493–497 (2017). https://doi.org/10.1038/nature22076

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